Etiologic Risk Group:NIH rating of Risk Group Level 3 for B.pseudomallei
*Disease Information:Burkholderia pseudomallei—a gram-negative bacterium—is the cause of a serious infectious disease known as melioidosis, or as Whitemore’s disease. Exposure to contaminated water or soil, followed by inhalation, ingestion, or contact with skin abrasions, accounts for most of the causes of transmission. Very few cases of human-to-human transfer of Melioidosis have been observed. Melioidosis is prominent in southeast Asia and in northern Australia, where it accounts for approximately 50% of sepsis-induced death. Current treatments are time and resource-laden, consisting of antimicrobial IV therapy requiring administration for 10-14 days in a hospital or clinical setting, followed by oral therapy required for 3-6 consecutive months. Yet this treatment regimen maintains high mortality rates at around 35% [1]. Treatment with more common antimicrobial agents, such as tetracyclines, macrolides, and fluoroquinolones is not effective due to B.pseudomallei’s ability to pump out such drugs.
Link to TDR Targets page (if present):none available
Essentiality of this protein:No essentiality data is available for 3UND in B.pseudomallei. However, it is reported that a homologous protein, 2-dehydro-3-deoxyphosphooctonate aldolase, found in the closely related sister species—B. thailandensis (gene BTH_I1893)—is essential [2]. The protein encoded by BTH_I1893 shows query coverage of 98%, maximum identity of 51%, and positives of 67% between the amino acid sequences of B.thailandensis and B.pseudomallei (see Figure 2 below). The high degree of homology observed in the primary amino acid sequence of each species’ aldolases provides support for comparison of protein essentiality across species. Additionally, B.thailandensis and B.pseudomallei are closely related species wherein B.thailandensis is often considered to be a less virulent strain of B.pseudomallei. For this reason, it is assumed that 3UND is also essential in B.pseudomallei. 3UND is involved in lipopolysaccharide biosynthesis, catalyzing the following reaction:
Figure 1- 3UND catalyzes the reaction between phosphoenolpyruvate and D-arabinose 5 phosphate, producing 2-dehydro-3-deoxy-D-octonate 8-phosphate and phosphate in the presence of water. Figure 2-A Basic Local Alignment Search Tool (BLAST) provided by NCBI was used to determine the homology betweenB.pseudomallei and B. thailandensis. The A chain from 2-dehydro-3-deoxyphosphooctonate aldolase of each organism was used.
Is it a monomer or multimer as biological unit?3UND is a tetrameter (multimer) as a biological unit.
Complex of proteins:3UND is a tetramer with identical A, B, C, and D subuntis of 281 amino acids per unit. However, structural analysis in PyMol indicates that even as a tetramer, A, B, C, and D possess individual active sites. Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):3UND is cited as having no “close” human homolog, making it a prime target for B.pseudomallei treatment. BRENDA documents more than 18 inhibitors for 3UND, however, only (Z,E)-D-Glucophosphoenolpyruvate has been found to specifically inhibit 3UND produced by/whithin gram-negative bacteria. (Note: Burkholderia pseudomallei is a gram-negative bacterium).
Enzyme Assay information: KDO8P (Aldolase) kinetic enzyme assay- “The standard assay was performed at 60C in a final volume of 200 ul reaction buffer consisting of 100 mM HEPES buffer pH 7.0, 0.48 mM Cd2+, 2 mM PEP, 2 mM A5P, 0.2 mM EDTA, and appropriately diluted enzyme (typically 0.3 uM). An aliquot of the enzyme (10 ul) was first preincubated with EDTA (10 ul, 4 mM) for 15 min at room temperature. To this solution was added the reaction buffer (160 ul) containing Cd2+ (0.48 mM) and the resulting mixture was incubated for 10 min at 60 C. The reaction was initiated by adding the mixture (20 ul) of both substrates, PEP (20 mM) and A5P (20 mM). After incubation at 60 C (typically for 10 min) the reaction was quenched by adding TCA to a final concentration of 5%.” -- link to page paper containing assay information: http://download-v2.springer.com/static/pdf/857/art%253A10.1007%252Fs00792-003-0346-3.pdf?token2=exp=1429370871~acl=%2Fstatic%2Fpdf%2F857%2Fart%25253A10.1007%25252Fs00792-003-0346-3.pdf*~hmac=e9b5a8a36606cba8c1d28647029e37adafd307e696a4caa082557ff3a7ecf2c8
Structure -- PDB #: 3UND http://www.rcsb.org/pdb/explore.do?structureId=3UND A BLASTP of the 3UND amino acid sequence compared to the human database of amino acids yielded “no significant similarity found” between 3UND and human proteins. While this finding could be an error, the lack of similarity is supported by the claim made by Baugh, Gallagher, Patrapuvich, et. al. that KDOP synthase lacks a close human homolog[1]. This finding suggests that targeting KDOP (3UND) may be a safe drug target. Figure 3-BLASTP homology search result between 3UND and the H.sapien protein databank. The A chain of 3UND was used (amino acid length: 281).
Current Inhibitors: According to BRENDA, the following ligands have been found as inhibitors to 3UND: (Z,E)-D-Glucophosphoenolpyruvate 1,10-phenanthroline, 1-carboxyheptane-1,7-diyl bis(phosphate) 2,6-Anhydro-3-deoxy-2beta-phosphonylmethyl-8-phosphate-D-glycero-D-talo-octonate 2,6-pyridine dicarboxylic acid 2-dehydro-3-deoxy-D-octonate 8-phosphate 2-Deoxy-2-fluoro-D-arabinoate-5-phosphate Bromopyruvate D-arabinose 5-phosphate dipicolinic acid EDTA However, (Z,E)-D-Glucophosphoenolpyruvate is the only ligand listed above that has been observed to bind to 3UND in gram negative bacteria. Six ligands have been shown to inhibit 3UND according to BindingDB, including: CHEMBL1938424 (56950652) CHEMBL95764 (3496) CHEMBL1941140 (57400865) CHEMBL1941138 (444348) CHEMBL1941139 (57393902) CHEMBL1235229 (5326965)
Expression Information:3UND is required for the synthesis of lipopolysaccharides, which are essential components of the outer membranes of gram-negative bacteria such as Burkholderia pseudomallei. KDOP synthetases are structurally conserved, and it has been anticipated that inhibitors to these enzymes will be effective across many species of Burkholderia, many of which are responsible for various infectious diseases, including cystic fibrosis infections and meliodosis infections [3]. Synthesis of biological membranes is a requirement of bacterial reproduction, meaning the inhibition of 3UND enzyme could prevent new membrane formation, thus preventing bacterial growth. In rapidly dividing B.pseudomallei—such as when bacteria are approaching log phase growth upon infecting a susceptible host—3UND would thus be expressed constitutively. The constitutive expression of BURPS1710b_3264, resulting in the synthesis of 3UND, suggests that a drug which targets 3UND may be highly effective in stopping infections. This is in contrast to non-constitutively transcribed genes, which are only transcribed and translated by organisms in the face of a specific environmental stimuli and would thus result in lower druggability due to more limited gene expression.
3UND has been successfully expressed in E.coli (K-12) [4].
Purification Method:It is possible to code for a 6 Histidine tag when engineering the BURPS1710b_3264 gene for transformation into an E.coli plasmid. The engineered N-terminus 6-His tag will then allow purification using Nickel-NTA column chromatography and FPLC. Other procedures, as documented on BRENDA, include “overnight dialysis against 50 mM ammonium acetate, pH 7.8, 4°C, reconstitution with substrates and products” http://www.brenda-enzymes.org/enzyme.php?ecno=2.5.1.55#Purification/COMMENTARY
Image of protein:
Figure 4- PyMol surface image of 3UND tetramer with blue A subunit carbons, green B subunit carbons, pink C subunit carbons, and yellow D subunit carbons. Oxygen atoms are shown in red and nitrogen atoms in blue.
Figure 5- PyMol cartoon of 3UND tetramer with blue A subunit, green B subunit, pink C subunit, and yellow D subunit. PEP shown as sticks colored by element with carbon as pink, oxygen as red, and phosphorous as orange. A5P shown as sticks colored by element with carbon as green, phosphate as orange, oxygen as red.
Figure 6- A subunit of 3UND shown as ribbon with protein carbons shown as green. A5P is shown as sticks with yellow carbon, red oxygen, and orange phosphorous. PEP is shown as sticks with pink carbon, red oxygen, and orange phosphorous.
Length of your protein in Amino Acids: 281 AA per chain, 1124 AA per tetramer Molecular weight of protein in kDa using Expasy ProtParam: 30150.9 kDa per chain Molar Extinction coefficient of protein at 280nm: 4845 M-1cm-1 for A subunit
TMpred graph Image:
Figure 7-TMpred Output of the “A” subunit of 3UND isolated from Burkholderia pseudomallei. Two possible transmembrane regions were identified with scores of 542 and 861. Inside to outside helices : 2 found from to score center 97 ( 99) 115 ( 115) 110 107 222 ( 222) 241 ( 238) 573 230
Outside to inside helices : 1 found from to score center 221 ( 221) 237 ( 237) 700 229
Figure 8-TMpred output for all four (A, B, C, and D) subunits of 3UND isolated from Burkholderia pseudomallei showed 8 possible transmembrane helices with significance scores ranging from 542-861, as outputted by TMpred, ExPASy.
*GC% Content for gene:66.1%; (559/846) bases *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): not needed now *GC% Content for gene (codon optimized): not needed now
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to. Primer design results for 'tail' primers (this is just 2 sequences): not needed now
References
[1] Northfield, J.; Whitty, C.; MacPhee, I. Burkholderia pseudomallei infection, or melioidosis, and nephrotic syndrome. Nephrol. Dial. Transplant. 2002, 17 (1), 137-139. doi:10.1093/ndt/17.1.137
[2] Baugh, L; Gallagher, L.A.; Patrapuvich R.; Clifton M.C. Combining Functional and Structural genomics to Sample the Essential Burkholderia Structome. PLoS ONE. 2013,** 8(1), 10033-10046.
*NCBI Gene # or RefSeq#: BURPS1710b_3264
*Protein ID #: 3UND
*Organism (including strain): Burkholderia pseudomallei 1710b
Etiologic Risk Group: NIH rating of Risk Group Level 3 for B.pseudomallei
*Disease Information: Burkholderia pseudomallei—a gram-negative bacterium—is the cause of a serious infectious disease known as melioidosis, or as Whitemore’s disease. Exposure to contaminated water or soil, followed by inhalation, ingestion, or contact with skin abrasions, accounts for most of the causes of transmission. Very few cases of human-to-human transfer of Melioidosis have been observed. Melioidosis is prominent in southeast Asia and in northern Australia, where it accounts for approximately 50% of sepsis-induced death. Current treatments are time and resource-laden, consisting of antimicrobial IV therapy requiring administration for 10-14 days in a hospital or clinical setting, followed by oral therapy required for 3-6 consecutive months. Yet this treatment regimen maintains high mortality rates at around 35% [1]. Treatment with more common antimicrobial agents, such as tetracyclines, macrolides, and fluoroquinolones is not effective due to B.pseudomallei’s ability to pump out such drugs.
Link to TDR Targets page (if present): none available
Link to Gene Database page:
https://www.patricbrc.org/portal/portal/patric/Feature?cType=feature&cId=PATRIC.271848.6.NC_007651.CDS.2141576.2142430.fwd
http://www.ncbi.nlm.nih.gov/gene/?term=Bth_I1893
Link to Protein Databases:
http://www.kegg.jp/dbget-bin/www_bget?bpm:BURPS1710b_3264
http://www.rcsb.org/pdb/explore.do?structureId=3UND
Essentiality of this protein: No essentiality data is available for 3UND in B.pseudomallei. However, it is reported that a homologous protein, 2-dehydro-3-deoxyphosphooctonate aldolase, found in the closely related sister species—B. thailandensis (gene BTH_I1893)—is essential [2]. The protein encoded by BTH_I1893 shows query coverage of 98%, maximum identity of 51%, and positives of 67% between the amino acid sequences of B.thailandensis and B.pseudomallei (see Figure 2 below). The high degree of homology observed in the primary amino acid sequence of each species’ aldolases provides support for comparison of protein essentiality across species. Additionally, B.thailandensis and B.pseudomallei are closely related species wherein B.thailandensis is often considered to be a less virulent strain of B.pseudomallei. For this reason, it is assumed that 3UND is also essential in B.pseudomallei. 3UND is involved in lipopolysaccharide biosynthesis, catalyzing the following reaction:
Figure 1- 3UND catalyzes the reaction between phosphoenolpyruvate and D-arabinose 5 phosphate, producing 2-dehydro-3-deoxy-D-octonate 8-phosphate and phosphate in the presence of water.
Figure 2- A Basic Local Alignment Search Tool (BLAST) provided by NCBI was used to determine the homology between B.pseudomallei and B. thailandensis. The A chain from 2-dehydro-3-deoxyphosphooctonate aldolase of each organism was used.
Is it a monomer or multimer as biological unit? 3UND is a tetrameter (multimer) as a biological unit.
Complex of proteins: 3UND is a tetramer with identical A, B, C, and D subuntis of 281 amino acids per unit. However, structural analysis in PyMol indicates that even as a tetramer, A, B, C, and D possess individual active sites.
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): 3UND is cited as having no “close” human homolog, making it a prime target for B.pseudomallei treatment. BRENDA documents more than 18 inhibitors for 3UND, however, only (Z,E)-D-Glucophosphoenolpyruvate has been found to specifically inhibit 3UND produced by/whithin gram-negative bacteria. (Note: Burkholderia pseudomallei is a gram-negative bacterium).
*EC#: 2.5.1.55
Link to BRENDA EC# page:
Link for B.pseudomallei-
http://www.kegg.jp/dbget-bin/www_bget?bpm:BURPS1710b_3264
Link for B.thailandensis-
http://www.genome.jp/dbget-bin/www_bget?bte:BTH_I1893
-- See above for screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information:
KDO8P (Aldolase) kinetic enzyme assay- “The standard assay was performed at 60C in a final volume of 200 ul reaction buffer consisting of 100 mM HEPES buffer pH 7.0, 0.48 mM Cd2+, 2 mM PEP, 2 mM A5P, 0.2 mM EDTA, and appropriately diluted enzyme (typically 0.3 uM). An aliquot of the enzyme (10 ul) was first preincubated with EDTA (10 ul, 4 mM) for 15 min at room temperature. To this solution was added the reaction buffer (160 ul) containing Cd2+ (0.48 mM) and the resulting mixture was incubated for 10 min at 60 C. The reaction was initiated by adding the mixture (20 ul) of both substrates, PEP (20 mM) and A5P (20 mM). After incubation at 60 C (typically for 10 min) the reaction was quenched by adding TCA to a final concentration of 5%.”
-- link to page paper containing assay information:
http://download-v2.springer.com/static/pdf/857/art%253A10.1007%252Fs00792-003-0346-3.pdf?token2=exp=1429370871~acl=%2Fstatic%2Fpdf%2F857%2Fart%25253A10.1007%25252Fs00792-003-0346-3.pdf*~hmac=e9b5a8a36606cba8c1d28647029e37adafd307e696a4caa082557ff3a7ecf2c8
-- links to assay reagents (substrates) pages.
A5P: http://www.sigmaaldrich.com/catalog/product/sigma/a2013?lang=en®ion=US
PEP: http://www.sigmaaldrich.com/catalog/product/aldrich/860077?lang=en®ion=US
--- List cost and quantity of substrate reagents, supplier, and catalog #
Phospho(enol)pyruvic acid monopotassium salt
Structure
-- PDB #: 3UND
http://www.rcsb.org/pdb/explore.do?structureId=3UND
A BLASTP of the 3UND amino acid sequence compared to the human database of amino acids yielded “no significant similarity found” between 3UND and human proteins. While this finding could be an error, the lack of similarity is supported by the claim made by Baugh, Gallagher, Patrapuvich, et. al. that KDOP synthase lacks a close human homolog[1]. This finding suggests that targeting KDOP (3UND) may be a safe drug target.
Figure 3- BLASTP homology search result between 3UND and the H.sapien protein databank. The A chain of 3UND was used (amino acid length: 281).
Current Inhibitors:
According to BRENDA, the following ligands have been found as inhibitors to 3UND:
(Z,E)-D-Glucophosphoenolpyruvate
1,10-phenanthroline,
1-carboxyheptane-1,7-diyl bis(phosphate)
2,6-Anhydro-3-deoxy-2beta-phosphonylmethyl-8-phosphate-D-glycero-D-talo-octonate
2,6-pyridine dicarboxylic acid
2-dehydro-3-deoxy-D-octonate 8-phosphate
2-Deoxy-2-fluoro-D-arabinoate-5-phosphate
Bromopyruvate
D-arabinose 5-phosphate
dipicolinic acid
EDTA
However, (Z,E)-D-Glucophosphoenolpyruvate is the only ligand listed above that has been observed to bind to 3UND in gram negative bacteria. Six ligands have been shown to inhibit 3UND according to BindingDB, including:
CHEMBL1938424 (56950652)
CHEMBL95764 (3496)
CHEMBL1941140 (57400865)
CHEMBL1941138 (444348)
CHEMBL1941139 (57393902)
CHEMBL1235229 (5326965)
Expression Information:3UND is required for the synthesis of lipopolysaccharides, which are essential components of the outer membranes of gram-negative bacteria such as Burkholderia pseudomallei. KDOP synthetases are structurally conserved, and it has been anticipated that inhibitors to these enzymes will be effective across many species of Burkholderia, many of which are responsible for various infectious diseases, including cystic fibrosis infections and meliodosis infections [3]. Synthesis of biological membranes is a requirement of bacterial reproduction, meaning the inhibition of 3UND enzyme could prevent new membrane formation, thus preventing bacterial growth. In rapidly dividing B.pseudomallei—such as when bacteria are approaching log phase growth upon infecting a susceptible host—3UND would thus be expressed constitutively. The constitutive expression of BURPS1710b_3264, resulting in the synthesis of 3UND, suggests that a drug which targets 3UND may be highly effective in stopping infections. This is in contrast to non-constitutively transcribed genes, which are only transcribed and translated by organisms in the face of a specific environmental stimuli and would thus result in lower druggability due to more limited gene expression.
3UND has been successfully expressed in E.coli (K-12) [4].
Purification Method: It is possible to code for a 6 Histidine tag when engineering the BURPS1710b_3264 gene for transformation into an E.coli plasmid. The engineered N-terminus 6-His tag will then allow purification using Nickel-NTA column chromatography and FPLC. Other procedures, as documented on BRENDA, include “overnight dialysis against 50 mM ammonium acetate, pH 7.8, 4°C, reconstitution with substrates and products”
http://www.brenda-enzymes.org/enzyme.php?ecno=2.5.1.55#Purification/COMMENTARY
Image of protein:
Figure 4- PyMol surface image of 3UND tetramer with blue A subunit carbons, green B subunit carbons, pink C subunit carbons, and yellow D subunit carbons. Oxygen atoms are shown in red and nitrogen atoms in blue.
Figure 5- PyMol cartoon of 3UND tetramer with blue A subunit, green B subunit, pink C subunit, and yellow D subunit. PEP shown as sticks colored by element with carbon as pink, oxygen as red, and phosphorous as orange. A5P shown as sticks colored by element with carbon as green, phosphate as orange, oxygen as red.
Figure 6- A subunit of 3UND shown as ribbon with protein carbons shown as green. A5P is shown as sticks with yellow carbon, red oxygen, and orange phosphorous. PEP is shown as sticks with pink carbon, red oxygen, and orange phosphorous.
*Amino Acid Sequence:
MNLAGFEVGLDKPFFLIAGTCVVESEQMTIDTAGRLKEICAKLGVPFIYKSSYDKANRSS
GKSFRGLGMDEGLRILAEVKRQLNVPVLTDVHEIDEIAPVAAVVDVLQTPAFLCRQTDFI
RACAQSGKPVNIKKGQFLAPHDMKNVIDKARDAAREAGLSEDRFMACERGVSFGYNNLVS
DMRSLAIMRETGAPVVFDATHSVQLPGGQGTSSGGQREFVPVLARAAVATGVAGLFMETH
PNPAEAKSDGPNAVPLGRMAALLETLVTLDQAVKRVPFLENDFN
Length of your protein in Amino Acids: 281 AA per chain, 1124 AA per tetramer
Molecular weight of protein in kDa using Expasy ProtParam: 30150.9 kDa per chain
Molar Extinction coefficient of protein at 280nm: 4845 M-1cm-1 for A subunit
TMpred graph Image:
Figure 7- TMpred Output of the “A” subunit of 3UND isolated from Burkholderia pseudomallei. Two possible transmembrane regions were identified with scores of 542 and 861.
Inside to outside helices : 2 found
from to score center
97 ( 99) 115 ( 115) 110 107
222 ( 222) 241 ( 238) 573 230
Outside to inside helices : 1 found
from to score center
221 ( 221) 237 ( 237) 700 229
Figure 8-TMpred output for all four (A, B, C, and D) subunits of 3UND isolated from Burkholderia pseudomallei showed 8 possible transmembrane helices with significance scores ranging from 542-861, as outputted by TMpred, ExPASy.
Inside to outside helices : 8 found
from to score center
8 ( 8) 27 ( 27) 861 17
226 ( 226) 244 ( 244) 542 236
290 ( 290) 312 ( 309) 904 300
511 ( 511) 529 ( 529) 542 521
575 ( 575) 597 ( 594) 904 585
796 ( 796) 814 ( 814) 542 806
860 ( 860) 882 ( 879) 904 870
1081 (1081)1099 (1099) 542 1091
Outside to inside helices : 8 found
from to score center
7 ( 7) 30 ( 25) 1148 17
223 ( 226) 244 ( 242) 672 234
292 ( 292) 315 ( 310) 1148 302
508 ( 511) 529 ( 527) 672 519
577 ( 577) 600 ( 595) 1148 587
793 ( 796) 814 ( 812) 672 804
862 ( 862) 885 ( 880) 1148 872
1078 (1081)1099 (1097) 672 1089
*CDS gene Sequence:
atgaacgtagcaatcagccccggcgtcacggccggcaacagcctgcctttcgtgctgttc
ggcgggatcaacgtgctcgagagtctcgacttcacgctcgacgtgtgcggcgaatacgtc
gcggtgacgcgcaagctcggcattccgttcgtgttcaaggcgtcgttcgacaaggcgaac
cgctcgtcgatccattcgtatcgcggcgtcgggctcgacgaaggcctgaagatcttcgcc
gaggtgaaggcgcgcttcggcgtgccggtgatcaccgatgtgcacgaagccgagcaggcg
gcgcccgtggccgaaatcgccgacgtgttgcaggtgcccgcgtttctcgcgcggcagacc
gatctcgtcgtcgcgatcgcgaaggccggcaagccggtgaacgtgaagaagccgcagttc
atgagccccacgcaattgaagcacgtggtgtcgaaatgcggcgaggtcggcaacgatcgc
gtgatgctgtgcgagcgcggcagttcgttcggctacgacaatctcgtcgtggacatgctc
ggcttccggcagatggccgagacgacgggcggttgcccggtgatcttcgacgtcacgcac
agcctgcagtgccgcgatccgctcggcgacgcgtcgggcggccggcgccggcaagtgctc
gatctcgcgcgcgcgggcatcgcggtcggcatcgcggggctctttctcgaggcgcacccc
gatcccgaccgcgcgcgctgcgatgggccgagcgcgctgccgttgcatcagctcgagggc
ttgctgtcgcagatgaaggcgatcgacgatctcgtcaagcgcatgccggcgctcgagatt
cgatga
*GC% Content for gene: 66.1%; (559/846) bases
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): not needed now
*GC% Content for gene (codon optimized): not needed now
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): not needed now
References
[1] Northfield, J.; Whitty, C.; MacPhee, I. Burkholderia pseudomallei infection, or melioidosis, and nephrotic syndrome. Nephrol. Dial. Transplant. 2002, 17 (1), 137-139. doi:10.1093/ndt/17.1.137
[2] Baugh, L; Gallagher, L.A.; Patrapuvich R.; Clifton M.C. Combining Functional and Structural genomics to Sample the Essential Burkholderia Structome. PLoS ONE. 2013,** 8(1), 10033-10046.
[3] http://www.ssgcid.org/target-status/featured-structures/kdop/
[4] http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/enzymes/GetPage.pl?ec_number=2.5.1.55